1. Field of the Invention
This invention relates to a method of catalyzing disulfide bond formation in fully reduced, cloned gene products produced in microbes such as Escherichia coli. More particularly, the invention concerns such a method of oxidation wherein the reaction is controlled to promote the in vitro formation of disulfide bridges which correspond to those present in the naturally occurring protein species.
2. Description of Related Disclosures
When native proteins which contain one or more disulfide bridges in their native state are produced as recombinant proteins in microorganisms, the protein produced is often in a reduced form, lacking disulfide bridges. In some cases the protein product may contain oligomers following purification. Such oligomers may be the result of uncontrolled oxidation or thiol-disulfide exchange reactions. If the native protein contains disulfide bonds, it will often be desirable to promote chemically the formation of the corresponding disulfide bonds in the recombinant protein product, while minimizing the formation of oligomers or other modified protein by-products. Oxidizing the protein in an uncontrolled manner may also result in the formation of undesirable isomers (incorrect intramolecular bridging). Such unwanted reactions may complicate the purification of the protein from the culture, reduce the yield of protein having the desired structure, or generate a protein with less than full bioactivity. In the case of certain proteins which are intended for therapeutic use, uncontrolled disulfide bond formation during purification or formulation may yield a nonhomogeneous material which is contaminated with isomers and/or oligomers which may be inactive and/or have increased immunogenicity.
U.S. Pat. No. 4,530,787 issued July 23, 1985 to Ze'ev Shaked et al., entitled "Controlled Oxidation of Microbially Produced Cysteine-Containing Proteins," describes a process for oxidizing such microbially produced proteins in a selective, contolled manner using a non-catalytic oxidizing agent, preferably o-iodosobenzoic acid, which oxidizes cysteines preferentially producing the desired disulfide bridging in high yield. This process requires at least stoichiometric amounts of oxidizing agent to protein to ensure that the oxidation proceeds to completion.
Similarly, a process has been described for the catalysis of disulfide bond formation in microbially produced rennet, using a mixture of oxidized and reduced glutathione in urea. (European patent application No. 83307841.3 published as European Publication No. 114,507 on Aug. 1, 1984 to Hayenga et al.)
It is known that ferricyanide or copper +2 ions are able to catalyze disulfide bond formation in .beta.-lactoglobulin in the presence of sodium dodecyl sulfate. Leslie, J. et al., Can. Jour. Biochem., 46, 625 (1968). Other disclosures teach use of specific divalent metal salts as oxidants for cysteine or the sulfhydryl groups in specific cases: (a) copper ion: (for free crysteine) Hanaki, A. et al., Bull Chem. Soc. Jpn., 56, 2065 (1983); (for sulfhydryls in lysozyme) Yutani, K. et al., J. Biochem., 64, 449 (1968); (for sulfhydryl compounds such as glutathione, cysteine, 2-mercaptoethanol, thioglycolic acid and reduced lipoic acid) Kobashi, K., Biochim. Biophys. Acta, 158, 239 (1968); (b) transition metals: (for cysteine and other mercaptans and proteins with free sulfhydryl groups) Friedman, Mendel, The Chemistry and Biochemistry of the Sulfhydryl Group in Amino Acids, Peptides and Proteins, (New York: Pergamon Press), Chapter 2, pp. 25-50 (1973); and (c) possibly calcium ion: (for sulfhydryls in deoxyribonuclease) Price, P. et al., J. Biol. Chem., 244, 929 (1969).
The mechanism of oxidation in these reactions is unclear, but has been postulated to be based on reactions involving peroxide or free radicals. However, it appears that the ability to predict that a given divalent salt will successfully promote the correct oxidation of a specific protein without extensive side reactions is not possible at this time. The present invention demonstrates the ability of certain metal-containing compounds to promote highly selective and useful formation of disulfides in various forms of recombinant interleukin-2 and .beta.-interferon.